小柯机器人

美国麻省理工学院-哈佛大学布罗德研究所David R. Liu团队研究利用双引物编辑实现了对长DNA序列进行可编程删除、替换、整合和反转。2021年12月9日出版的《自然-生物技术》杂志发表了这项成果。

研究人员研发了双引物编辑(twinPE)，这是一种不依赖于DNA双链断裂 (DSB)的方法，它使用一个引物编辑器蛋白和两个引物编辑引导RNA (pegRNA) 来对内源性人类基因组位点的DNA序列进行可编程替换或切除。这两个pegRNA以基因组DNA反义链上互补的DNA瓣为模板，这些DNA瓣取代了引物编辑器诱导切口位点之间的内源性DNA序列。

当与位点特异性丝氨酸重组酶结合时，twinPE能够在人类细胞中靶向整合基因大小的DNA质粒 (>5,000 bp) 和40 kb的靶向序列倒置。TwinPE扩展了精准基因编辑的能力，并可能与其他工具协同作用，以纠正或补充大型或复杂的人致病等位基因。

据介绍，基因组序列的靶向缺失、替换、整合或倒位可用于研究或治疗人类遗传疾病，但现有方法通常需要DSB，这会导致严重副作用，包括未知的indel混合物和染色体异常。

附：英文原文

Title: Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Author: Anzalone, Andrew V., Gao, Xin D., Podracky, Christopher J., Nelson, Andrew T., Koblan, Luke W., Raguram, Aditya, Levy, Jonathan M., Mercer, Jaron A. M., Liu, David R.

Issue&Volume: 2021-12-09

Abstract: The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000bp) and targeted sequence inversions of 40kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

DOI: 10.1038/s41587-021-01133-w

Source: https://www.nature.com/articles/s41587-021-01133-w

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex